Fluorescent bioprobes have made important contributions to advancing our knowledge in life science, thanks to their unrivalled ability to image and monitor biological structures and processes in living systems. Typical materials used as biosensors include natural polymers, inorganic nanoparticles, and organic dyes. Green fluorescent protein (GFP), for example, has been used as a reporter of expression for morphological differentiation. The biosensing process, however, requires complicated and time-consuming transfection procedures, which can lead to unexpected morphologies and undesired abnormality in the target cells. Inorganic nanoparticles, such as quantum dots (QDs), are highly luminescent and resistant to photobleaching but limited in variety and inherently toxic to living cells, because QDs are commonly made of heavy metals and chalcogens (e.g., CdSe and PbS).
Organic dyes are rich in variety and have been widely used as readily processable light-emitting materials, particularly in the area of organic optoelectronics. Due to their poor miscibility with water, organic dyes are prone to aggregate in aqueous media, which normally weakens their light emissions. This effect is commonly known as aggregation-caused quenching (ACQ). Incorporation of cationic (e.g., aminium) or anionic (e.g., sulfonate) groups into organic dyes can improve their miscibility with physiological buffer. The charged species repel each other and prevent the dye molecules from aggregating, hence alleviating the ACQ effect.
Although the ionic groups can help enhance their solubility in water, the dyes are still inclined to aggregate at high concentrations due to the hydrophobicity of their aromatic cores. This is likely why dyes are commonly used in only trace amounts (often at the nM level). At high concentrations, the electric charges of the ionized dyes may affect membrane potentials, perturb intracellular physiology and even cause cell lyses. On the other hand, at low dye concentrations, the fluorescence signals are weak and the small amount of dye molecules that enter cells are easily photobleached during the imaging process. Furthermore, during cell division and when confluent cells are passed, the intracellular dye molecules may diffuse back to the extracellular media due to the concentration gradient. This results in a decrease in the fluorescence of the stained cells and a concurrent increase in the solution fluorescence (or background emission) as well as random staining in co-culture systems of different types of cell lines.
One way to keep the dye molecules inside cells and to prevent them from leaking to media is to fix them through bioconjugation. This necessitates further attachment of new reactive groups to the ionic dyes. Since the bioconjugations are usually conducted in situ under physiological conditions, the reactions are normally incomplete, wherein the extent of chemical transformation is unknown. This makes it difficult to learn how and to what extent the reactions affect the metabolism and physiology of the living cells stained by the conjugation processes. The unconjugated dyes can escape from the cells, and even the conjugated dyes can be released back to the culture media, because the biomolecular units responsible for the bioconjugation may be degraded by the enzymatic reactions in the incubation processes and by the phototoxic effects of the bioimaging process.
Accordingly, there is a great need for the development of fluorescent bioprobes with high biological compatibility, strong photobleaching resistance, efficient light emission, and that are nontoxic to live cells, and have the ability to stay inside live cells for a long period of time without leaking out into the culture media.